Metallopharmaceutical diagnostic and therapeutic agents are finding more and more applications in biological and medical researches, and in diagnostic and therapeutic processes. In general, these agents contain radioisotopes or paramagnetic metals, and, when introduced into a subject, are accumulated at the selected specific organ, tissue or lesion. For diagnostic purposes, the in vivo distribution of the radioisotopes or paramagnetic metals may be imaged in various ways. The distribution of the detected radioisotopes or paramagnetic metals and their relative intensity can elucidate the space occupied by the targeted tissue as well as the presence of receptors and antigens, anomalies, pathological conditions, or the like. In therapeutic applications, the agents tend to contain radioisotopes, and the radiopharmaceuticals deliver a specific dose of radiation to the disease site.
Depending on the targeted organ or tissue of interest and on the desired diagnostic or therapeutic procedures, a range of metallopharmaceuticals can be used. One common type is a conjugate in which a carrier that transports the conjugate to a specific target organ or tissue site is chemically attached to a radioactive or paramagnetic metal. The metal is usually coordinated with the conjugate and, more commonly, is linked in the form of a macrocyclic chelate (For example, refer to Liu's U.S. Pat. No. 6,916,460.).
The Cu-64 radionuclide has attracted special interests in nuclear medicine and imaging, owing to its half-life (t1/2=12.7 h), decay properties (β+ 19%; (β− 39%) and large-scale producibility with high specific activity using a biomedical cyclotron, and hence the potential in positron emission tomography (PET) and targeted radiotherapy. In this regard, one of the most important things is to prepare the radionuclide into stable complexes using various chelating agents, so that they can be delivered to specific/targeted tissues without transmetalation from conjugates to biomolecules. In vivo conditions, there are many circumstances where the kinetic stability of Cu(II) is more important than its thermodynamic stability. The N-acetic acid pendant arm consisting mainly of cyclen and cyclam and its derivatives have been studied as bifunctional chelators (BFCs). DOTA and TETA, which are Cu(II) macrocyclic chelators derived from cyclen and cyclam, have shown comparable or better kinetic and biological stabilities as compared to acyclic chelators such as EDTA or DTPA. However, researches on biodistribution and metabolism of 64Cu-DOTA and 64Cu-TETA revealed in vivo instability due to transchelation of 64Cu as well as accumulation at the liver and high absorption in non-specific tissues resulting therefrom. The applicability of these chelators as radiopharmaceutical is determined by their flexibility, pore size, ability and extent of complexation, separation kinetics of the complex, or the like. In order to improve the in vivo stability of the metal complexes, transchelation and non-specific absorption and accumulation should be reduced. For this, chemical modification of the backbone of this type of polyazamacrocycles has been reported. That is to say, side-bridges and cross-bridges are introduced to improve stability of complexes.
1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) is widely used as a BFC for various copper and other radionuclides for clinical contrast agents and peptide- and antibody-targeted radiation therapies. However, according to a recent study, TETA is not an ideal BFC because of dissociation of the metal from the chelate and binding with proteins. Anderson et al. have experimentally demonstrated the dissociation of 64Cu from TETA-D-Phe1-octreotide and its binding with superoxide dismutase (SOD). Weisman and Wong have designed the new-concept ethylene cross-bridged azamacrocyclic chelator CB-TE2A with two acetate pendant arms replaced by ethylene bridge between two non-adjacent nitrogens of TETA. The introduction of 1,8-ethylene cross-bridge in CB-TE2A resulted in improved stability of the metal complex while providing structural rigidity. Anderson et al. also have synthesized and described ethylene cross-bridged CB-DO2A Cu(II) complexes. These cross-bridged ligands, the octahedrally coordinated Cu(II) complexes of CB-TE2A and CB-DO2A, are enclosed by the four nitrogen electron pairs of chelators and the two carboxylate pendant arms to give Cu(II)-CB-TE2A and Cu(II)-CB-DO2A. Research showed that the new cross-bridged ligands were able to form kinetically more stable Cu(II) complexes and biologically more stable radiolabeled complexes when compared to non-cross-bridged DO2A or TETA. The 64Cu-CB-TE2A-Tyr3-octreotate resulted in faster clearance in blood, liver and kidneys as compared to other similar 64Cu-TETA derivatives.
In order to prevent the possibility of one carboxylate groups being consumed for conjugation, another pendant arm may be covalently bonded to further improve in vivo stability. Lewis et al. have synthesized a CB-TE2A derivative in which a biotin molecule is covalently attached. However, the kinetic and in vivo stability of the Cu(II)-CB-TE2A-Bz-biotin is not known. Boswell et al. have synthesized cross-bridged TE2A (CB-TE2A), which is similar to CB-TE2A in structure but has a third orthogonally protected arm allowing conjugation with peptides and other targeting agents regardeless of the hexacoordination position of Cu(II). Although in vivo experiment was not carried out, in vitro test revealed that the radiolabeled peptide conjugate is a stable radiocopper complex without new transchelation with human serum proteins for 48 hours.
WO 02-26267 and US Patent Publication No. 2006-62728 disclose ethylene cross-bridged tetraaza macrocyclic compounds and uses thereof. More specifically, US Patent Publication No. 2006-62728 discloses a compound represented by the following chemical formula, which is cross-bridged by C2 ethylene between two nitrogens. However, the ethylene cross-bridged tetraaza macrocyclic compound is disadvantageous in that a metal complex is formed by coordination with a metal element only at high temperatures, because the cross-bridging aliphatic hydrocarbon is short. The ethylene cross-bridged tetraaza macrocyclic compound is prepared by reacting cyclam or cyclen with glyoxal (CHO—CHO). However, since a functional group cannot be attached to glyoxal, it is impossible to attach a functional group such as NCS to the cross-bridged ethylene, that can bind with a bioactive substance (J. Am. Chem. Soc. 2000, 122, 10561-10572). Accordingly, the only option is to attach the bioactive substance to the functional group bound to the nitrogen atom (e.g., COOH or NCS in the following chemical formula). However, when the bioactive substance is attached to the functional group bound to the nitrogen atom, it is difficult for the functional group to serve as a ligand upon coordination with a metal ion. As a result, the resulting coordination compound has lower in vivo stability and activity.

Although the existing ethylene cross-bridged tetraaza macrocyclic compound has good in vivo stability, it requires very high temperature (80-100° C.) to form a coordination complex with a metal element. Accordingly, when it is to be conjugated with a bioactive substance or a chemically active substance (For example, the NCS moiety in the above chemical formula is bound to the bioactive substance or chemically active substance.), the bioactive substance or the chemically active substance can be damaged (e.g., denaturation of protein), making it inapplicable as a therapeutic agent or a diagnostic agent.
For the tetraaza macrocyclic compound to be actually utilized, for example, as a contrast agent for nuclear imaging, it has to form a complex with a radioactive metal element. In this case, because of the short half-life of the radioactive metal element, the actual product is produced as a tetraaza macrocyclic compound with a bioactive substance attached thereto (For products with no bioactive substance attached, the procedure of attaching the bioactive substance has to be carried out in the hospital. But, this is almost impossible except for some special cases. Hence, all the products are sold in the form with the bioactive substance attached to the tetraaza macrocyclic compound.). That is to say, the hospitals purchase the tetraaza macrocyclic compound with the bioactive substance already attached thereto and form a complex with a radioactive metal element for use as a contrast agent or a radioactive therapeutic agent. Accordingly, if the temperature required for the complex formation with the metal ion is high, the bioactive substance (e.g., protein) bound thereto may be denatured. For this reason, the existing ethylene cross-bridged tetraaza macrocyclic compound is not commercially applicable in many cases.